Closely tied to the concept of a "standard" endotoxin is the idea of a TPD for such a standard endotoxin. The establishment of a defined, specific endotoxic level has allowed the concept to be established that a certain amount of endotoxin is allowable and a certain amount of endotoxin should not be delivered into the bloodstream or cerebrospinal fluid. The advent of LAL allowed the quantitation of endotoxin as a contaminant. In turn, quantitation has allowed for the creation of specific and relevant endotoxin limits for manufactured drug products, raw materials, active ingredients, devices, components, depyrogenation processes, and in-process samples that constitute the legal requirement for releasing to market products that are not considered "adulterated" by the US FDA.
Today's user of the LAL test rightly views such concepts as the bread and butter of endotoxin testing, but it is good to appreciate the degree to which today's system of endotoxin quantitation has progressed, in that
1. "Quantification" in the rabbit assay was limited to a pass/fail response (rabbit response = 0.6°C temperature rise);
2. The pyrogen test was initially established without attempting to quantify the amount of endotoxin necessary to produce a febrile response;
3. Early LAL testing used the weight of dried bacterial endotoxins in nanograms first with various gram-negative organisms, then with a specific strain of E. coli.
None of the early tests could have been used effectively to develop product specific tolerance limits (TLs) as they exist today, much less provide the degree of in-process control needed for modern pharmaceutical and biotechnology manufacturing contamination control. In some respects, the 10- to 1000fold greater sensitivity of the LAL test created the "luxury" of controversy on several fronts. A whole new system of relating the new assay to the existing test had to be developed to avoid unnecessary product test failures due to the greater sensitivity of the LAL assay (43). The "system" included the formation of or association with:
1. The EU as a measure of relative biological activity
2. The TL (endotoxin limit concentration)
3. The maximum valid dilution to relate the product dose to the allowable endo-toxin content (realizing that a positive LAL response in any given solution as in the pyrogen assay would be inappropriately stringent)
4. The lysate sensitivity (lambda) to standardize the relative reactivity of each LAL to each control standard endotoxin (CSE) (Fig. 2)
Prior to this "system" several of the principals of the early LAL assay expressed concern that the greater sensitivity of the assay would end up becoming an apparent disadvantage used by some to confound industry efforts to develop the assay as a replacement for the rabbit pyrogen test. ["I hope that we do not turn the advantage provided by the greater sensitivity of the Limulus test into a problem"— Jack Levin (21)].
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